Discoverx Corporation

Leland Stanford Junior University

Kalobios, Inc.

Thomas S Wehrman

William Raab

Helen M Blau

Keith R Olson

Chin Yee Loh

Daniel Bassoni

Jeng-Horng Her

Robert F Balint

Wei Feng

Philip Achacoso

Mahesh Mathrubutham

Methods and materials are disclosed for use in an enzyme fragment complementation assay using complementary fragments of &#x3b2;-galactosidase to study the trafficking of proteins in a cell. Compounds that bind to a target peptide have been found to affect protein folding and therefore trafficking. &#x3b2;-Galactosidase fragments, an enzyme donor (ED) and an enzyme acceptor (EA), are fused to a target peptide and to an intracellular compartment protein, wherein the compartment is involved in intracellular trafficking. Contacting the cell with a compound that binds to the target peptide results in enhanced movement of the protein through the cellular trafficking pathway comprised of the endoplasmic reticulum, Golgi apparatus, the plasma membrane, endosomes, etc. Using this approach, compounds that bind to a target peptide and alter its ability to traffic through the normal cellular pathway can be readily detected.

Monitoring protein trafficking using beta-galactosidase reporter fragment complementation

Methods and compositions are provided to measure the binding of a test compound to a target peptide by measuring the effect of the compound on the abundance of the target peptide inside a cell. The target peptide may bind the test compound at an active site or an allosteric site, and it has been found that such binding may stabilize the target peptide against cellular degradation. The target peptide will preferably comprise a destabilizing mutation which shortens the half life of the target peptide within the cell, typically a mammalian cell. Test compounds, including small molecules, have been found to stabilize target peptides. Also provided are systems and kits for use in practicing the methods.

Detection of intracellular binding events by measuring protein abundance

Methods and genetic constructs are provided for detecting the binding of nuclear hormone receptors to a coactivator/corepressor. The methods employ enzyme fragment complementation using fragments of &#x3b2;-galactosidase as the detection system. Cells are transformed to express the large fragment of &#x3b2;-galactosidase fused to a member of the complex with NHR for initiation of transcription and have it localized in the nucleus and to express the small fragment of &#x3b2;-galactosidase fused to the nuclear hormone receptor for binding to the member upon stimulation with a ligand.

Assays for nuclear hormone receptor binding

Methods and compositions are provided for detecting molecular translocations, particularly protein translocations within and between sub-cellular compartments, using at least two components that exhibit a localization-dependent difference in complementation activity. In particular, alpha-complementing &#x3b2;-galactosidase fragments are provided. These &#x3b2;-galactosidase reporter fragments display significantly enhanced enzymatic activity when one fragment is localized in a membrane. Methods for carrying out no-wash ELISA assays based on the reporter component system are also provided.

Detection of protein translocation by beta-galactosidase reporter fragment complementation

Methods and compositions are provided for detecting molecular translocations, particularly protein translocations within and between subcellular copartments, using at least two components that exhibit a localization-dependent difference in complementation activity. In particular, alpha-complementing &#x3b2;-galactosidase fragments are provided. These &#x3b2;-galactosidase reporter fragments display significantly enhanced enzymatic activity when one fragment is localized in a membrane. Methods for carrying out no-wash ELISA assays based on the reporter component system are also provided.

A method for determining ligand activation of receptors using cells expressing genetic constructs of a fusion protein of at least a binding domain of an auxiliary protein and a fragment of &#x3b2;-galactosidase, a fusion protein of an endosome-associated protein and a complementary fragment of &#x3b2;-galactosidase, and a wild-type receptor. The receptors are characterized by binding to the auxiliary protein-binding domain upon activation by an agonist and then endocytosing associated with an endosome to which the endosome-associated protein binds. Cells are incubated with a candidate ligand followed by lysis with a lysing medium comprising a substrate for the &#x3b2;-galactosidase. The enzyme product is then detected as a measure of the activation of the receptor.

Wild-type receptor assays

Methods and compositions for detecting molecular interactions, particularly protein-protein interactions, using at least two inactive, weakly-complementing &#x3b2;-lactamase fragments are provided. The invention allows detection of such interactions in eukaryotic and mammalian cells, in situ or in vitro. Detection of molecular interactions in mammalian cells is not limited to the nuclear compartment, but can be accomplished in the cytoplasm, cell surface, organelles, or between these entities. Methods provided utilize novel compositions comprising fusion proteins between molecules of interest and inactive, weakly-complementing &#x3b2;-lactamase fragments. Association of the molecules of interest brings the corresponding complementary &#x3b2;-lactamase fragments into close enough proximity for complementation to occur and &#x3b2;-lactamase activity to be observed. The invention is useful in the study of protein-protein interactions, functional genomics, agonist and antagonist screening and drug discovery.

Detection of molecular interactions by &#x3b2;-lactamase reporter fragment complementation

Truncated fragments of the small fragment of &#x3b2;-galactosidase are provided that have low affinity for the large fragment of &#x3b2;-galactosidase and provide for robust signals when two fusion proteins are complexed due to the binding of the proteins to which the &#x3b2;-galactosidase fragments are fused. The truncated fragments do not interfere with the complexing of the two proteins and allow for the two proteins to function and be responsive to candidate compounds that affect complex formation.

&#x3b2;-galactosidase donor fragments

Methods for detecting phosphorylation of receptor tyrosine kinases ('RTKs') upon activation are provided. The method employs cells comprising two fusion products: (1) an RTK fused to a small fragment of &#x3b2;-galactosidase and (2) a phosphotyrosine binding peptide fused to the large fragment of &#x3b2;-galactosidase, where the 2 fragments weakly complex to form an active enzyme, and optionally a construct for a cytosolic RTK phosphorylating kinase, when the RTK does not autophosphoryate. To detect phosphorylation a &#x3b2;-galactosidase substrate is added to the cells, whereby product formation indicates the occurrence of phosphorylation.

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Mountain View, CA, United States of America

Thomas S Wehrman